This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Specific Aims. Aim 1. Characterize the rate at which SFG rickettsiae spread through individual ticks and throughout individual tick tissues. Uninfected Dermacentor variabilis will be exposed to Rickettsia rickettsii, R. montanensis, or R. bellii via capillary feeding. Parameters of SFG infection and transmission, including dispersion and multiplication, in individual tick tissues will be determined. A. Assess SFG rickettsial infection and subsequent tissue dissemination in the tick vector using quantitative real-time PCR. B. Quantify rickettsiae associated with horizontal (salivary gland) and vertical (ovary) transmission events in infected egg, larva, nymph, and adult stages. Aim 2. Identify and characterize uniquely expressed protein from three tick-borne rickettsiae in tick hosts during horizontal and vertical transmission events. Although not well understood, a metabolic union between other SFG rickettsiae and their tick hosts has been described. Rickettsia undergo a reactivation event changing in cell structure and vertebrate infectivity in association with host environmental cues coupled to bloodmeal acquisition. This aim will provide novel information concerning tick-borne rickettsiae in relation to global protein expression and antigenic profiles. A. Initial studies will compare protein profiles of SFG rickettsiae during transmission events within the tick tissues involved in horizontal (salivary gland) and vertical (ovary) transmission by two dimensional gel electrophoresis. B. Identify rickettsial antigens by immunoblot using polyclonal antibodies to tick-borne rickettsiae and liquid chromatography quadruple-time-of-flight (2DLC-QTOF) analysis. C. Compare observed protein expression to gene expression, as assessed by real-time PCR.